A large variety of expression systems has been described in the patent as well as in the scientific literature. However, despite the fact that fusion proteins have become a cornerstone of modern biology, obtaining the target protein in a soluble, biologically active form, as well as in high yield, continues to be a major challenge (Kapust, R. B. and Waugh, D. S., Protein Sci 8 (1999) 1668-74).
Examples of fusion partners that have been touted as solubilizing agents include thioredoxin (TRX), glutathione S-transferase (GST), maltose-binding protein (MBP), Protein A, ubiquitin, and DsbA. Although widely recognized and potentially of great importance, this solubilizing effect remains poorly understood. It is not clear, for example, what characteristics besides intrinsically high solubility epitomize an effective solubilizing agent. Are all soluble fusion partners equally proficient at this task, or are some consistently more effective than others? Similarly, it is not known whether the solubility of many different polypeptides can be improved by fusing them to a highly soluble partner, or whether this approach is only effective in a small fraction of cases.
The state of the art relating to the most potent expression systems has recently been summarized by Kapust et al., supra. In their attempt to produce soluble fusion proteins comprising various target proteins they assessed three different and prominent candidate fusion partners. Maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX) were tested for their ability to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. All these candidate expression systems are known to the skilled artisan and described in detail elsewhere (e.g., EP 293 249 describes in detail the use of GST as an expression tool).
Remarkably, Kapust et al., supra, found that MBP is a far more effective solubilizing agent than the other two fusion partners also widely used in the art. Moreover, they demonstrated that only in some cases, fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation.
It is especially critical that many aggregation-prone polypeptides may be rendered soluble by fusing them to an appropriate partner, but that some candidate fusion partners in a more or less unpredictable way are much better solubilizing agents than others.
A great demand therefore exists to provide for alternative, efficient expression tools, which are especially appropriate for the recombinant expression of aggregation prone proteins, e.g. like the rsgps.
There is a wealth of patent literature relating to proteins which bind to the immunosupressant FK-506, the so-called FK-506 binding proteins or FKBPs.
These proteins have been extensively studied and commercial applications have been designed centering around the FK-506 binding activity of these proteins. For example, WO 93/25533 makes use of CTP:CMP-3-deoxy-D-manno-octulosonate cytidyl transferase (=CKS) as expression tool. A FKBP is inserted into a CKS-based expression vector down-stream of the CKS gene. The fusion protein obtained is used to improve measurements of FK-506 and other immunosuppressants.
WO 00/28011 discloses materials and methods for regulation of biological events such as target gene transcription and growth, proliferation and differentiation of engineered cells.
WO 97/10253 relates to a high throughput assay for screening of compounds capable of binding to a fusion protein which consists of a target protein and an FK-506-binding protein. Disclosed is the use of an FKBP12-Src homology (SH2) fusion protein in an high throughput screening assay. The fusion protein is produced in soluble form in the bacterial periplasm and released by standard freeze-thaw treatment.